Project Summary/Abstract: Around half of allergic asthmatics are classified as Th2-high (vs. Th2-low) endotype due to increased type 2 inflammation as compared with healthy controls. Airway type 2 inflammation, characterized by increased Th2 (type 2) cytokines (IL-4, IL-5, IL-9, IL-13), is mediated by interplays between multiple cell types of the lung, including structural cells (e.g. epithelial cells), and infiltrating cells (e.g. Th2 cells, group 2 innate lymphoid cells (ILC2s)). Although most of Th2-high asthmatics are responsive to current steroid-based therapy, a notable subgroup of patients with this endotype show steroid-resistance and require high-dose steroids to control their persistent symptoms. Therefore, there is an unmet need for developing novel steroid-sparing therapy to reduce the side effects associated with high-dose use of steroids. Interleukin (IL)-25 (also called IL-17E) has been known as an important upstream regulator of the classic Th2 response. The single-nucleotide polymorphism (SNP) of IL-25 receptor (IL-25R) subunit IL-17RB has been linked to risk of asthma, implicating that targeting IL- 25 may be of clinical significance in a subset of asthmatics. We previously identified Act1 (TRAF3IP2) as an essential adaptor molecule in IL-25 signaling and established its critical role in IL-25-mediated allergic pulmonary inflammation. Excitingly, we recently found that TNF receptor associated factor 4 (TRAF4) was a novel signaling molecule meditating Act1 recruitment to IL-25R, exerting a critical functional role for IL-25- mediated airway inflammation. The RING domain of TRAF4 protein is a structural feature that confers protein with E3 ligase activity. We and others have shown that TRAF4 is a genuine E3 ligase which induces polyubiquitination and the ubiquitination of its binding partners through its RING finger domain. We thus hypothesize that TRAF4 functions as an E3 ligase in IL-25 signaling impacting both Act1-dependent and - independent pathways in a cell-specific manner to promote type 2 allergic asthma. To test this hypothesis, we propose the following Specific Aims: 1) Investigate the intrinsic role of TRAF4 in different cell compartments in type 2 allergic asthma; 2) Delineate the molecular mechanism of TRAF4-mediated IL- 25 signaling. The proposed in-depth cellular and molecular studies on the TRAF4 E3 ligase in IL-25 signaling will provide significant insight into the events that initiate and maintain the asthmatic phenotype. Moreover, the results from our proposed studies may improve the potential to identify new drug targets and develop novel steroid-sparing drugs (e.g pathway-specific decoy peptides targeting IL-25 signaling) for treatment of asthma.